Computational protocol: Modulation of Network Oscillatory Activity and GABAergic Synaptic Transmission by CB1 Cannabinoid Receptors in the Rat Medial Entorhinal Cortex

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[…] Combined EC-hippocampal slices were prepared from young male Wistar rats (50–110 g) as previously described []. All experiments were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and European Communities Council Directive 1986 (86/609/EEC). Rats were anaesthetised with isoflurane and N2/O2, until cardiorespiratory arrest, and decapitated. The brain was rapidly removed and immersed in oxygenated artificial cerebrospinal fluid (ACSF) chilled to 4°C. Slices (450 μm) were cut using a vibrating microtome (MicroM, Germany), and stored in ACSF continuously bubbled with 95% O2/5% CO2, at room temperature. Following a recovery period of at least 1 hour, individual slices were transferred to a recording chamber mounted on the stage of an Olympus (BX50WI) upright microscope. The chamber was continuously perfused with oxygenated ACSF at 30–32°C, at a flow rate of approximately 2 mL/min. The ACSF contained the following (in mM): NaCl (126), KCl (3.25), NaH2PO4 (1.25), NaHCO3 (24), MgSO4 (2), CaCl2 (2.5), and D-glucose (10). The solution was continuously bubbled with 95% O2/5% CO2 to maintain a pH of 7.4. Neurones were visualized using differential interference contrast optics and an infrared video camera.Patch-clamp electrodes were pulled from borosilicate glass (1.2 mm OD, 0.69 ID; Harvard Apparatus) and had open tip resistances of 4-5 MΩ. They were filled with a solution containing the following (in mM): CsCl (90), HEPES (33), QX-314 (5), EGTA (0.6), MgCl2 (5.0), TEA-Cl (10), phosophocreatine (7) ATP (4), GTP (0.4). The solution was adjusted to 290 mOsmol with sucrose and to pH 7.4 with CsOH. Whole-cell voltage clamp recordings were made from neurones in layers II and V of the medial division of the EC, using an Axopatch 700 series amplifier (Molecular Devices, USA). The holding potential in all cases was –70 mV. Under these experimental conditions, layer II/V neurones exhibited sIPSCs, mediated by GABA acting primarily at GABAA receptors.Data were recorded directly to computer hard disk using AxoScope software (Molecular Devices, USA). Mini Analysis (Synaptosoft, USA) was used for analysis of sIPSCs offline. sIPSCs were detected automatically using a threshold-crossing algorithm, and their frequency and amplitude are analysed. 200 sIPSCs were sampled during a continuous recording period for each neurone under each condition. The nonparametric Kolmogorov-Smirnoff (KS) test was used to assess the significance of shifts in cumulative probability distributions of interevent interval (IEI). Differences between drug and control situations in studies of sIPSCs were assessed by means of a one-way ANOVA. All error values stated in the text refer to the S.E.M.All salts used in preparation of ACSF were Analar grade and purchased from Merck/BDH (UK). LY320135 and ACPA were obtained from Tocris Cookson (UK).For field recordings of oscillatory activity, slices were placed into an interface chamber (BRSC-1, Digitimer, UK) and the chamber was continuously perfused with oxygenated ACSF at 30–32°C, at a flow rate of approximately 1-2 mL/min. Extracellular population recordings were made with glass microelectrodes filled with aCSF, of resistance 1–3 MΩ. Signals were amplified 1000-fold and recorded unfiltered. Low amplitude 50 Hz interference was removed using a HumBug (Quest Scientific, Canada). Signals were digitized and recorded at 10 kHz using an NPI EXT-02F amplifier (NPI, Germany) and pClamp 10 software (Molecular Devices, USA). Following 30–90 minutes control period of stable oscillatory activity, drugs were applied to the bath in known concentration. Pharmacological oscillatory activity was analysed using the fast Fourier transform (FFT, Clampfit 10), cross-correlation analysis and Morlet-wavelet time-frequency spectrogram analysis (MatLab 2007R, Mathworks). Student t-tests were carried out to determine statistical significance.We analysed oscillations at beta (15–29 Hz) and gamma (30–90 Hz) bands, using bandpass filters (Clampfit 10.1) and measurement of the area under the power spectrum curve in Sigmaplot 8.0. […]

Pipeline specifications

Software tools pCLAMP, SigmaPlot
Applications Miscellaneous, Patch-clamp
Organisms Rattus norvegicus